1. Field of the Invention
This invention is drawn to transgenic plants with improved resistance to Fusarium head blight and nucleic acid constructs used in their production.
2. Description of the Prior Art
Wheat (Triticum aestivum) is fundamental to the world's food supply. Worldwide, one of the most serious threats to wheat production is Fusarium head blight (FHB) (Windels. 2000. Economic and social impacts of Fusarium head blight: changing farms and rural communities in the northern great plains. Phytopathology. 90:17-21; Dean et al. 2012. The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol. 13:414-430), which is caused when Fusarium graminearum (Schwabe) (teleomorph: Gibberella zeae) infects the spike. FHB, also called “scab”, results in significant yield loss and grain contaminated with the mycotoxin deoxynivalenol (DON). Losses for the United States wheat and barley harvests from 1998 to 2000 have been estimated to total $2.7 billion dollars (Wood et al. 1999. Fighting Fusarium. In Agricultural Research. United States Department of Agriculture, Agricultural Research Service, Beltsville, Md.). Aside from yield reduction, contamination of grain with DON is a serious health problem as it can cause illness in both humans and animals. The US Food and Drug Administration has advised that DON concentration in products not exceed 1 ppm for humans and 5 ppm for livestock (Van Egmond and Dekker. 1995. Worldwide regulations for mycotoxins in 1994. Natural Toxins. 3:332-336). Grain presented at storage elevators is tested for DON content, and producers are assessed price penalties for higher levels of contamination. Unfortunately FHB is becoming more frequent; in large part because the F. graminearum is able to grow saprophytically on the debris of corn. As corn acreage is expanding rapidly and the adoption of conservation tillage is increasing, wheat is frequently planted into soil with an ample source of FHB inoculum (Bai and Shaner. 2004. Management and resistance in wheat and barley to Fusarium head blight. Annual Review of Phytopathology. 42:135-161).
FHB can occur anywhere in the world where rainfall or high humidity occurs during flowering (Steffenson. 2003. Fusarium head blight of barley: impact, epidemics and strategies for identifying and utilizing genetic resistance. In L K J, B W R, eds, Fusarium Head Blight of Wheat and Barley. APS Press, St. Paul, Minn., pp 241-295; Bai and Shaner. 2004. ibid.). Airborne F. graminearum spores are thought to enter the spikelet through openings in the palea and lemma tissue. Once it has entered the spikelet the fungus initially grows without killing cells, although it is debated whether this represents a true biotrophic phase as intracellular growth has not been observed during this initial phase of infection (Bushnell et al. 2003. Histology and physiology of Fusarium head blight. In K Leonard, W R Bushnell, eds, Fusarium head blight of wheat and barley. APS Press, St. Paul, Minn., pp 44-83; Jansen et al. 2005. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proceedings of the National Academy of Sciences of the United States of America. 102:16892-16897; Trail. 2009. For blighted waves of grain: Fusarium graminearum in the postgenomics era. Plant physiology. 149:103-110; Brown et al. 2010. The infection biology of Fusarium graminearum: defining the pathways of spikelet to spikelet colonisation in wheat ears. Fungal Biol. 114:555-571). However, within hours the pathogen transitions to a clearly necrotrophic phase accompanied by rapid growth, intracellular invasion and the biosynthesis of DON, which is essential for spreading through the rachis and into neighboring spikelets (Bai et al. 2001. Deoxynivalenol-nonproducing Fusarium graminearum causes initial infection, but does not cause disease spread in wheat spikes. Mycopathologia. 153:91-98; Jansen et al. 2005. ibid).
Intense efforts have been made by wheat breeders to develop FHB resistant varieties, but none of these have strong resistance, as all identified sources of genetic resistance are Quantitative Trait Loci (QTL) that provide only partial protection (Mesterhazy. 2003. Breeding wheat for Fusarium head blight resistance in Europe. In K Leonard, W R Bushnell, eds, Fusarium head blight of wheat and barley. APS Press, St. Paul, Minn., pp 211-240; Bai and Shaner. 2004. ibid). In wheat the most widely utilized QTL is FHB1, located on chromosome 3BS (Anderson et al. 2008. Toward positional cloning of Fhb1, a major QTL for Fusarium head blight resistance in wheat. Cereal Research Communications. 36:195-201). Plants containing the FHB1 allele have what is defined as type II resistance or resistance to spread of infection, but not to initial infection (Schroeder and Christensen. 1963. Factors affecting resistance of wheat to scab caused by Giberella zeae. Phytopathology. 53:831-838). This type of resistance is assayed by point inoculating one spikelet and then observing whether infection spreads through the rachis and into neighboring spikelets.
Despite clear contribution of these loci to FHB resistance, little is known about the genes residing at the QTL or about the mechanisms of resistance. One of the reasons this research has been so difficult in wheat is the complexity of performing genetic analysis in an allohexaploid species. Given the difficulty of genetic analysis several groups have begun to study the FHB resistance response by characterizing the transcriptional changes that occur in resistant and susceptible interactions (Boddu et al. 2006. Transcriptome analysis of the barley-Fusarium graminearum interaction. Mol Plant Microbe Interact. 19:407-417; Bernardo et al. 2007. Fusarium graminearum-induced changes in gene expression between Fusarium head blight resistant and susceptible wheat cultivars. Funct Integr Genomics. 7:69-77; Boddu et al. 2007. Transcriptome analysis of trichothecenes-induced gene expression in barley. Mol Plant Microbe Interact. 20:1364-1375; Golkari et al. 2007. Microarray analysis of Fusarium graminearum-induced wheat gene: identification of organ-specific and differentially expressed genes. Plant Biotechnology Journal. 5:38-49; Kong et al. 2007. Expression analysis of defense-related genes in wheat in response to infection by Fusarium graminearum. Genome. 50:1038-1048; Desmond et al. 2008. Gene expression analysis of the wheat response to infection by Fusarium pseudograminearum. Physiological and Molecular Plant Pathology. 73:40-47; Li and Yen. 2008. Jasmonate and ethylene signaling pathway may mediate Fusarium head blight resistance in wheat. Crop Science. 48:1888-1895; Jia et al. 2009. Transcriptome analysis of a wheat near-isogenic line carrying Fusarium head blight-resistant and -susceptible alleles. MPMI. 1366-1378; Steiner et al. 2009. Differential gene expression of related wheat lines with contrasting levels of head blight resistance after Fusarium graminearum inoculation. Theor Appl Genet. 118:753-764; Ding et al. 2011. Resistance to hemi-biotrophic F. graminearum infection is associated with coordinated expression of diverse defense signaling pathways. PLoS One. 6:e19008; Walter and Doohan. 2011. Transcript profiling of the phytotoxic response of wheat to the Fusarium mycotoxin deoxynivalenol. Mycotoxin Research. 1-10). This analysis has pointed toward a key role for induction of the basal defense pathway, also known as PAMP-triggered immunity (PTI). PTI is one of the two main branches of active plant immune responses. It is triggered by the perception of a range of conserved pathogen-associated molecular patterns by the host's PAMP perception recognition receptors (PRRs).
Several studies examining gene expression changes during FHB have detected induction of the ethylene (ET)- and jasmonic acid (JA)-signaling pathways as key responses of wheat as it is challenged by F. graminearum (Li and Yen, 2008. ibid; Ding et al., 2011. ibid). The ET- and JA-signaling pathways are integral components of the basal defense response and are known to synergistically activate basal defense against a wide range of necrotrophic pathogens (Glazebrook. 2005. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annual Review of Phytopathology. 43:205-227; Mengiste. 2012. Plant Immunity to Necrotrophs. Annu Rev Phytopathol.). ET-mediated responses, the subject of this report, are made up of ET-biosynthesis, perception, and signaling. ET-biosynthesis is up-regulated rapidly after many pathogens initiate infection. Perception of this gaseous hormone by ET-receptors results in the induction of a diverse array of ET-responsive transcription factors (ERFs), some of which have defense-specific functions. Defense components induced by ERFs include the PRR, FLS2 and BIK1, a receptor-kinase that supports the function of several PRRs, including FLS2, EFR and CERK1. Additional cellular and physiological changes associated with ET-signaling include induction of lignin synthesis (Mengiste. 2012. ibid) and formation of abscission zones. The study of Ding et al. (2011. ibid), which examined transcriptional and proteomic changes occurring at the very early stages of susceptible and resistant FHB interactions identified a possibly critical role for ET-signaling. Their analysis found a biphasic pattern of defense pathway expression. Within the first three hours of infection of susceptible and resistant genotypes, which likely corresponds to the biotrophic or asymptomatic phase of F. graminearum infection, elements of salicylic acid-mediated defense are induced. The second phase was defined as beginning at 12 hours after infection when genes associated with jasmonic acid-mediated defense are induced. However, it was observed that unique to the resistant genotype, genes involved in ET-biosynthesis and signaling are induced as early as 3-6 hours after infection. Ding et al suggested that activation of ET-signaling prior to JA-mediated defense in the resistant genotype is necessary for the JA-mediated defenses to have full activity.
However, Chen et al. (2009. Fusarium graminearum exploits ethylene signaling to colonize dicotyledonous and monocotyledonous plants. New Phytol. 182:975-983) proposed a very different role for ET-signaling in the FHB interaction. These authors developed an Arabidopsis-based model pathosystem for FHB in which F. graminearum infects detached leaves. Analysis of the behavior of Arabidopsis ET-signaling mutants in this pathosystem suggested that mutations eliminating ET-signaling conferred increased resistance to F. graminearum. They then conducted experiments in wheat that led them to conclude that F. graminearum exploits ET-signaling to create susceptibility to FHB.
Despite these and other advances, the need remains for plants with improved resistance to FHB.